Rapid, high-throughput, culture-based PCR methods to analyze samples for viable spores of Bacillus anthracis and its surrogates

Journal of Microbiological Methods
S R KaneE Raber

Abstract

To rapidly remediate facilities after a biothreat agent release, improved turnaround times are needed for sample analysis. Current methods to confirm the presence of a viable biothreat agent are limited by low sample throughput. We have developed a rapid-viability-polymerase chain reaction (RV-PCR) method to determine the presence of viable spores. The method combines high-throughput sample processing with 96-well PCR analysis, which measures a change in real-time, quantitative PCR response arising from increased target-cell populations during culturing. The method accurately detects 1 to 10 live spores in a high-dead spore background (10(6)). Field tests using approximately 1000 biological indicators, each containing 10(6) spores of the B. anthracis surrogate, Bacillus atrophaeus, exposed to seven lethal and sub-lethal chlorine dioxide levels showed no significant difference (p>0.05) between RV-PCR and standard culturing methods for detecting the percent survival of spores. RV-PCR results were obtained in <17 h compared to 7 days for the standard culturing method. High-throughput sample processing and RV-PCR protocols were also developed and tested for synthetic wipe samples containing reference dirt material. RV-PCR protocols...Continue Reading

References

May 23, 2002·Risk Analysis : an Official Publication of the Society for Risk Analysis·Ellen RaberPete Estacio
Dec 9, 2003·International Journal of Environmental Health Research·Ellen RaberKenneth Bogen
Jun 6, 2006·Applied and Environmental Microbiology·L R HodgesM J Arduino

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Citations

Aug 6, 2013·Applied Microbiology and Biotechnology·Bidyut R Mohapatra, Myron T La Duc
May 5, 2010·Antimicrobial Agents and Chemotherapy·Linda M WeigelSegaran P Pillai
Jul 19, 2011·Applied and Environmental Microbiology·Sonia E LétantSanjiv R Shah
Sep 20, 2011·Applied and Environmental Microbiology·Sheila Van CuykKristin M Omberg
Nov 26, 2010·Investigative Genetics·David L GreenbergDavid M Wagner
Apr 15, 2016·Biomolecular Detection and Quantification·S M Da SilvaJ B Morrow
Jan 16, 2013·Journal of Microbiological Methods·E Marielle RemillardKristin M Omberg
Mar 22, 2014·Frontiers in Microbiology·Ekaterina OsmekhinaPeter Neubauer
Sep 5, 2013·The Journal of Physical Chemistry. B·Michal Kurzynski, Przemyslaw Chelminiak
Jun 27, 2009·Journal of Medical Microbiology·Geraint B RogersKenneth D Bruce
Jul 30, 2021·Journal of Microbiological Methods·Rebecca N BushonVicente Gallardo

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